basic principles and applications of electrophoresis
DESCRIPTION
Order Form for Electrophoresis Kits and Reagents Please click this sentence to download the formTRANSCRIPT
![Page 1: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/1.jpg)
電泳
![Page 2: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/2.jpg)
Theory of ElectrophoresisThe movement of a charged molecule subjected to an electric field is represented by the following equation:
V = Eqf
V: the velocity of the molecule
E: the electric field in volts/cm
q: the net charge on the molecule
f: frictional coefficient, which depend on the mass and shape of the molecule
![Page 3: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/3.jpg)
Gel Electrophoresis of DNA
Agarose slab gel submerged in buffer
Wells for sample loading
Cathode (-)
Direction for DNA migration Anode (+)
Agarose is a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution. When an electric field is applied to an agarose gel in the presence of a buffer solution which will conduct electricity, DNA fragments move through the gel towards the positive electrode (DNA is highly negatively charged) at a rate which is dependent on its size and shape.
![Page 4: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/4.jpg)
![Page 5: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/5.jpg)
![Page 6: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/6.jpg)
Gel Electrophoresis of DNA
For linear DNA molecules, they have uniform shape and charge to mass ratio. The electrophoretic mobility of the DNA molecule is influenced primarily by the molecular size: The larger molecules are retarded by the molecular sieving effect of the gel, and the small molecules have greater mobility.
![Page 7: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/7.jpg)
Gel Electrophoresis of DNA
• The DNA can be stained by the inclusion of ethidium bromide in the gel, or by soaking the gel in a solution of ethidium bromide after electrophoresis. The DNA shows up as an orange band on illumination by UV light. Alternatively, methylene blue can be used to stain DNA.
• Gels composed of polyacrylamide can separate DNA molecules that differ in length by only one nucleotide and are used to determine the base sequence of DNA. Agarose gels are used to separate DNA fragments that have larger size differences.
![Page 8: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/8.jpg)
Digestion of DNA by Restriction Enzymes
Before the electrophoresis, DNA is digested by restriction enzymes into small fragments of DNA.
GAATTCCTTAAG
G AATTCCTTAA G
EcoRI
![Page 9: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/9.jpg)
Restriction enzymes - enzymes isolated from bacteria that cut DNA at specific sites(restriction sites)EcoRI - 5'- G A A T T C -3‘
3'- C T T A A G -5'
Properties of DNA-Double helixBuilding block(dA, dC, dG and dT)negatively charged at neutral pHAT and GC complementary pairing
![Page 10: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/10.jpg)
Baterial plasmid DNAPlasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome.They: are small (a few thousand base pairs)usually carry only one or a few genes are circularhave a single origin of replication
![Page 11: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/11.jpg)
+
BglI 2166
BglI 472
EcoRI 701
Plasmid DNA for digestion – pBluescript II SK+
![Page 12: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/12.jpg)
Agarose
A linear polymer extracted from seaweed
Migration of DNA in agarose dependent on four factors
- molecular size of the DNA- agarose concentration- conformation of the DNA- applied current
![Page 13: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/13.jpg)
wells
Anode(+)
Cathode(-)
1.5% agarosegel stained with methylene blue
DNA fragments of different sizes
![Page 14: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/14.jpg)
Wells
Bromophenol blue
Xylene cyanol FF
Tracking dye
![Page 15: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/15.jpg)
Methylene blue staining» to visualize the DNA fragments, stain agarose gel overnight with 1X methylene blue staining solution» safe alternative for DNA staining» easy available» non-carcinogenic
DNA fragments
![Page 16: Basic Principles and Applications of Electrophoresis](https://reader035.vdocuments.us/reader035/viewer/2022062317/5a4d1b8c7f8b9ab0599bf390/html5/thumbnails/16.jpg)
Base pairs distance migrated(mm)
A B C D M
316 47
656 38
1808 231634 25
DNA fragments of different sizes
DNA fragments of known sizes